Analogues of pyy

ABSTRACT

Analogues of PYY differing from native human PYY by substitution of Ser23 with Ala23, Glu23, Lys23, Gln23 or AIB23. Further optional features include substitutions at further positions, loss of the Tyrl residue of native human PYY and amidation of the C-terminus. Suitable for use as pharmaceuticals for treating and preventing disorders, in particular diabetes and obesity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Application No. 16/769,760,filed Jun. 4, 2020, now U.S. Pat. No. 11,421,012, issued Aug. 23, 2022,which is the National Stage of International Application No.PCT/GB2018/053513, filed Dec. 4, 2018, which claims the benefit of andpriority to Great Britain Patent Application No. 1720188.0, filed Dec.4, 2017, the contents of which are incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Sep. 22, 2022, is namedP024947US1_Sequence_listing_ST26.xml, and is 63,340 bytes in size.

FIELD OF THE INVENTION

This application relates to analogues of peptide YY (PYY), which areuseful in treating disorders such as diabetes and obesity, either aloneor in combination with other agents, especially in combination withGLP-1 analogues.

BACKGROUND OF THE INVENTION

According to the National Health and Nutrition Examination Survey(NHANES, 2011 to 2012), over two thirds of adults in the United Statesare overweight or obese. In the United States, 78% percent of males and74% percent of women, of the age of 20 or older, are either overweightor obese. In addition, a large percentage of children in the UnitedStates are overweight or obese.

The cause of obesity is complex and multi-factorial. Increasing evidencesuggests that obesity is not a simple problem of self-control but is acomplex disorder involving appetite regulation and energy metabolism. Inaddition, obesity is associated with a variety of conditions associatedwith increased morbidity and mortality in a population. Although theetiology of obesity is not definitively established, genetic, metabolic,biochemical, cultural and psychosocial factors are believed tocontribute. In general, obesity has been described as a condition inwhich excess body fat puts an individual at a health risk.

There is strong evidence that obesity is associated with increasedmorbidity and mortality. Disease risk, such as cardiovascular diseaserisk and type-2 diabetes disease risk, increases independently withincreased body mass index (BMI). Indeed, this risk has been quantifiedas a five percent increase in the risk of cardiac disease for females,and a seven percent increase in the risk of cardiac disease for males,for each point of a BMI greater than 24.9 (see Kenchaiah et al., N.Engl. J. Med. 347:305, 2002; Massie, N. Engl. J. Med. 347:358, 2002). Inaddition, there is substantial evidence that weight loss in obesepersons reduces important disease risk factors. Even a small weightloss, such as 10% of the initial body weight in both overweight andobese adults has been associated with a decrease in risk factors such ashypertension, hyperlipidemia, and hyperglycemia.

Although diet and exercise provide a simple process to decrease weightgain, overweight and obese individuals often cannot sufficiently controlthese factors to effectively lose weight. Pharmacotherapy is available;several weight loss drugs have been approved by the Food and DrugAdministration that can be used as part of a comprehensive weight lossprogram. However, many of these drugs have serious adverse side effects.When less invasive methods have failed, and the patient is at high riskfor obesity related morbidity or mortality, weight loss surgery is anoption in carefully selected patients with clinically severe obesity.However, these treatments are high-risk, and suitable for use in only alimited number of patients. It is not only obese subjects who wish tolose weight. People with weight within the recommended range, forexample, in the upper part of the recommended range, may wish to reducetheir weight, to bring it closer to the ideal weight. Thus, a needremains for agents that can be used to effect weight loss in overweightand obese subjects.

PYY is a 36-amino acid peptide produced by the L cells of the gut, withhighest concentrations found in the large bowel and the rectum. Twoendogenous forms, PYY and PYY 3-36, are released into the circulation.PYY 3-36 is further produced by cleavage of the Tyr-Pro amino terminalresidues of PYY by the enzyme dipeptidyl peptidase IV (DPP-IV). PYY 3-36binds to the Y2 receptor of the Y family of receptors (De Silva andBloom, Gut Liver, 2012, 6, p10-20). Studies have shown that peripheraladministration of PYY 3-36 to rodents and humans leads to markedinhibition of food intake, leading to the prospect that analogues of PYYmay be useful in treating conditions such as obesity (see, e.g.Batterham et al, Nature, 2002, 418, p650-654; Batterham et al, NewEngland Journal of Medicine, 2003, 349, p941-948).

PYY has also been implicated in altering the metabolism of subjects andhas been proposed as a treatment for type-2 diabetes, following evidencethat it is able to restore impaired insulin and glucagon secretion intype-2 diabetes. The relationship between obesity and diabetes iscomplex because being overweight increases diabetic risk and beingdiabetic increases the likelihood of being overweight. The nexus betweenthe two conditions is one in which PYY plays an increasingly recognizedrole.

WO2011/092473 and WO2012/101413 (Imperial Innovations Limited) disclosecertain analogues of PYY. However, there remains a need for furthercompounds which have suitable properties so that they are effective astherapeutic agents in treating or preventing disorders of energymetabolism such as obesity and/or diabetes.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an analogue of PYY whichdiffers from the sequence of native human PYY (Tyr1 Pro2 Ile3 Lys4 Pro5Glu6 Ala7 Pro8 Gly9 Glu10 Asp11 Ala12 Ser13 Pro14 Glu15 Glu16 Leu17Asn18 Arg19 Tyr20 Tyr21 Ala22 Ser23 Leu24 Arg25 His26 Tyr27 Leu28 Asn29Leu30 Val31 Thr32 Arg33 Gln34 Arg35 Tyr36; SEQ ID NO:1) in the followingrespect:

-   Ser23 is substituted with Ala23, Glu23, Lys23, Gln23 or AIB23    (preferably Ala23) and which may further differ from the sequence of    native human PYY in one of more of the following further respects:-   Tyr1 is absent,-   one or more of residues 2 to 9, 13 to 16, 20, 21, 24, 25 or 26 are    subject to a conservative substitution,-   Glu10 is substituted with Ala10, LyslO or Gln10-   Asp11 is substituted with Gly11-   Ala12 is substituted with AIB 12-   Leu17 is substituted with Ile17 or AIB17-   Asn18 is substituted with Leu18, AIB 18 or Ala18-   Arg19 is substituted with His19 or Lys19-   Ala22 is substituted with Val22 or Ile22-   Tyr27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31

-   and wherein the C-terminal residue optionally terminates in a    primary amide (—C(O)NH₂) group in place of a carboxylic acid group    (—CO₂H);-   or a derivative of the compound; or a salt of the compound or the    derivative.

The present invention is based on the discovery that analogues of PYY inwhich specific amino acid residues are deleted and/or substituted canalso be administered to a subject in order to cause decreased foodintake, decreased caloric intake, decreased appetite and an alterationin energy metabolism. In many cases the PYY analogues of the presentinvention exhibit improved potency and/or longer duration of actionand/or fewer side effects than native PYY.

The PYY analogues of the present invention are also especially suitablefor use in combination therapies with agonists of the GLP-1 receptor.This is because PYY and GLP-1 analogues have broadly compatible andsimilar chemistries which lend them to being formulated in combination,so they can be conveniently administered as a single injection.Additionally, PYY analogues and GLP-1 analogues inhibit appetite bydifferent and separate mechanisms, and so a patient receiving acombination therapy is less liable to ‘escape’ the desiredpharmaceutical effect than would be the case if treated with eitheragent alone. Lastly, the different mechanisms of action allow for anadditive or synergistic effect on appetite suppression, making a morepotent therapy

In another aspect, the invention provides a pharmaceutical compositioncomprising an analogue of PYY according to the invention together with apharmaceutically acceptable carrier and optionally a further therapeuticagent.

In another aspect, the invention provides an analogue of PYY accordingto the invention, or a pharmaceutical composition comprising the PYYanalogue, for use as a medicament. The analogue of PYY or pharmaceuticalcomposition find use in the prevention or treatment of a disorder ofenergy metabolism such as diabetes and/or obesity, for use in preventingloss of pancreatic islet function and/or for use in recoveringpancreatic islet function, in a subject.

In another aspect, the invention provides use of an analogue of PYYaccording to the invention for the manufacture of a medicament for theprevention or treatment of a disorder of energy metabolism such asdiabetes and/or obesity, for preventing loss of pancreatic isletfunction and/or for recovering pancreatic islet function, in a subject.

In another aspect, the invention provides a method of treating orpreventing a disease or disorder or other non-desired physiologicalstate in a subject, comprising administering a therapeutically effectiveamount of an analogue of PYY according to the invention, or apharmaceutical composition comprising the PYY analogue, to the subject.The analogue of PYY or pharmaceutical composition find use in methodsfor preventing or treating a disorder of energy metabolism such asdiabetes and/or obesity, preventing loss of pancreatic islet functionand/or recovering pancreatic islet function, reducing appetite, reducingfood intake, and/or reducing calorie intake in a subject.

In another aspect, the invention also provides a method of causingweight loss or preventing weight gain in a subject for cosmeticpurposes, comprising administering an effective amount of an analogue ofPYY according to the invention, or a composition comprising the PYYanalogue, to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are tables listing the amino acid sequences of some PYYanalogues that relate to specific preferred embodiments of theinvention. The naturally occurring sequence of human PYY is included forreference. The first column headed “REF” gives the SEQ ID NO: used inthe accompanying sequence listing. Native human PYY is REF = 1.

FIG. 2 shows the results of an in vitro receptor potency experimentdescribed in the examples

FIGS. 3A-3D show the results of an in vivo experiment (n = 5 to 8)demonstrating efficacy of compound Y242 as described in the examples.

FIGS. 4A-4D show the results of an in vivo experiment (n = 7 to 8)demonstrating efficacy of compound Y1419 as described in the examples.

FIGS. 5A-5D show the results of an in vivo experiment demonstratingefficacy of other compounds of the invention

FIGS. 6A-6T show the results of an in vitro receptor potency experimentdescribed in the examples

SEQUENCE LISTING

The amino acid sequences listed in the application are shown usingstandard letter abbreviations for amino acids. The specific sequencesgiven herein relate to specific preferred embodiments of the invention.The application comprises a machine-readable sequence listing whereinPYY analogues are allocated SEQ ID NO identifiers which are the same asthe Example numbers given in FIGS. 1A-1D.

DEFINITIONS

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Appetite: A natural desire, or longing for food. In one embodiment,appetite is measured by a survey to assess the desire for food.Increased appetite generally leads to increased feeding behavior.

Appetite Suppressants: Compounds that decrease the desire for food.Commercially available appetite suppressants include, but are notlimited to, amfepramone (diethylpropion), phentermine, mazindol,phenylpropanolamine fenfluramine, dexfenfluramine, and fluoxetine.

Body Mass Index (BMI): A mathematical formula for measuring body mass,also sometimes called Quetelet’s Index. BMI is calculated by dividingweight (in kg) by height² (in meters²). The current standards for bothmen and women accepted as “normal” are a BMI of 20-24.9 kg/m². In oneembodiment, a BMI of greater than 25 kg/m² can be used to identify anobese subject. Grade I obesity corresponds to a BMI of 25-29.9 kg/m².Grade II obesity corresponds to a BMI of 30-40 kg/m²; and Grade IIIobesity corresponds to a BMI greater than 40 kg/m² (Jequier, Am. J Clin.Nutr. 45:1035-47, 1987). Ideal body weight will vary among species andindividuals based on height, body build, bone structure, and sex.

Conservative substitutions: The replacement of an amino acid residue byanother, biologically similar residue in a polypeptide. The term“conservative variation” also includes the use of a substituted aminoacid, i.e. an amino acid with one or more atoms replaced with anotheratom or group, in place of a parent amino acid provided that thepolypeptide retains its activity or provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide. Typical but not limiting conservative substitutions are thereplacements, for one another, among the aliphatic amino acids Ala, Val,Leu and Ile; interchange of hydroxyl-containing residues Ser and Thr,interchange of the acidic residues Asp and Glu, interchange between theamide-containing residues Asn and Gln, interchange of the basic residuesLys and Arg, interchange of the aromatic residues Phe and Tyr, andinterchange of the small-sized amino acids Ala, Ser, Thr, Met and Gly.Additional conservative substitutions include the replacement of anamino acid by another of similar spatial or steric configuration, forexample the interchange of Asn for Asp, or Gln for Glu.

Non-limiting examples of conservative amino acid substitutions

Original Residue Conservative Substitutions Ala Gly, Val, Leu, Ile, Ser,Thr, Met Arg Lys Asn Asp, Gln, His Asp Glu, Asn Cys Ser Gln Asn, His,Lys, Glu Glu Asp, Gln Gly Ala, Ser, Thr, Met His Asn, Gln Ile Ala, Leu,Val, Met Leu Ala, Ile, Val, Met, Lys Arg Met Leu, Ile, Ala, Ser, Thr,Gly Phe Leu, Tyr, Trp Ser Thr, Cys, Ala, Met, Gly Thr Ser, Ala, Ser,Met, Gly Trp Tyr, Phe Tyr Trp; Phe Val Ala, Ile, Leu

Non-conservative substitutions: The replacement, in a polypeptide, of anamino acid residue by another residue which is not biologically similar.For example, the replacement of an amino acid residue with anotherresidue that has a substantially different charge, a substantiallydifferent hydrophobicity or a substantially different spatial or stericconfiguration.

Diabetes: A failure of cells to transport endogenous glucose acrosstheir membranes either because of an endogenous deficiency of insulinand/or a defect in insulin sensitivity. Diabetes is a chronic syndromeof impaired carbohydrate, protein, and fat metabolism owing toinsufficient secretion of insulin or to target tissue insulinresistance. It occurs in two major forms: insulin-dependent diabetesmellitus (IDDM, type I) and non-insulin dependent diabetes mellitus(NIDDM, type II) which differ in etiology, pathology, genetics, age ofonset, and treatment.

The two major forms of diabetes are both characterized by an inabilityto deliver insulin in an amount and with the precise timing that isneeded for control of glucose homeostasis. Diabetes type I, or insulindependent diabetes mellitus (IDDM) is caused by the destruction of βcells, which results in insufficient levels of endogenous insulin.Diabetes type II, or non-insulin dependent diabetes, results from adefect in both the body’s sensitivity to insulin, and a relativedeficiency in insulin production.

Food intake: The amount of food consumed by an individual. Food intakecan be measured by volume or by weight. For example, food intake may bethe total amount of food consumed by an individual. Or, food intake maybe the amount of proteins, fat, carbohydrates, cholesterol, vitamins,minerals, or any other food component, of the individual. “Proteinintake” refers to the amount of protein consumed by an individual.Similarly, “fat intake,” “carbohydrate intake,” “cholesterol intake,”“vitamin intake,” and “mineral intake” refer to the amount of proteins,fat, carbohydrates, cholesterol, vitamins, or minerals consumed by anindividual.

Normal Daily Diet: The average food intake for an individual of a givenspecies. A normal daily diet can be expressed in terms of caloricintake, protein intake, carbohydrate intake, and/or fat intake. A normaldaily diet in humans generally comprises the following: about 2,000,about 2,400, or about 2,800 to significantly more calories. In addition,a normal daily diet in humans generally includes about 12 g to about 45g of protein, about 120 g to about 610 g of carbohydrate, and about 11 gto about 90 g of fat. A low calorie diet would be no more than about85%, and preferably no more than about 70%, of the normal caloric intakeof a human individual.

In animals, the caloric and nutrient requirements vary depending on thespecies and size of the animal. For example, in cats, the total caloricintake per pound, as well as the percent distribution of protein,carbohydrate and fat varies with the age of the cat and the reproductivestate. A general guideline for cats, however, is 40 cal/lb/day (18.2cal/kg/day). About 30% to about 40% should be protein, about 7% to about10% should be from carbohydrate, and about 50% to about 62.5% should bederived from fat intake. One of skill in the art can readily identifythe normal daily diet of an individual of any species.

Obesity: A condition in which excess body fat may put a person at healthrisk (see Barlow and Dietz, Pediatrics 102:E29, 1998; NationalInstitutes of Health, National Heart, Lung, and Blood Institute (NHLBI),Obes. Res. 6 (suppl. 2):51S-209S, 1998). Excess body fat is a result ofan imbalance of energy intake and energy expenditure. For example, theBody Mass Index (BMI) may be used to assess obesity. In one commonlyused convention, a BMI of 25.0 kg/m² to 29.9 kg/m² is overweight, whilea BMI of 30 kg/m² or greater is obese.

In another convention, waist circumference is used to assess obesity. Inthis convention, in men a waist circumference of 102 cm or more isconsidered obese, while in women a waist circumference of 89 cm or moreis considered obese. Strong evidence shows that obesity affects both themorbidity and mortality of individuals. For example, an obese individualis at increased risk for heart disease, non-insulin dependent (type-2)diabetes, hypertension, stroke, cancer (e.g. endometrial, breast,prostate, and colon cancer), dyslipidemia, gall bladder disease, sleepapnea, reduced fertility, and osteoarthritis, amongst others (seeLyznicki et al., Am. Fam. Phys. 63:2185, 2001).

Overweight: An individual who weighs more than their ideal body weight.An overweight individual can be obese, but is not necessarily obese. Forexample, an overweight individual is any individual who desires todecrease their weight. In one convention, an overweight individual is anindividual with a BMI of 25.0 kg/m² to 29.9 kg/m².

Pegylated and pegylation: the process of reacting a poly(alkyleneglycol), preferably an activated poly(alkylene glycol) to form acovalent bond. A facilitator may be used, for example an amino acid,e.g. lysine. Although “pegylation” is often carried out usingpoly(ethylene glycol) or derivatives thereof, such as methoxypoly(ethylene glycol), the term is not limited herein to the use ofmethoxy poly(ethylene glycol) but also includes the use of any otheruseful poly(alkylene glycol), for example poly(propylene glycol).

pI: pI is an abbreviation for isoelectric point. An alternativeabbreviation sometimes used is IEP. It is the pH at which a particularmolecule carries no net electric charge. At a pH below its pI a proteinor peptide carries a net positive charge. At a pH above its pI a proteinor peptide carries a net negative charge. Proteins and peptides can beseparated according to their isoelectric points using a technique calledisoelectric focussing which is an electrophoretic method that utilises apH gradient contained within a polyacrylimide gel.

Peptide YY (PYY): The term PYY as used herein refers to a peptide YYpolypeptide, a hormone secreted into the blood by cells lining the lowersmall intestine (the ileum) and the colon. Naturally occurring wild typePYY sequences for various species are shown in Table 1.

TABLE 1 PYY sequence of various species PEPTIDE YY AA SEQUENCE HumanYPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO:1) Human 3-36IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO:2) Rat (Rattus norvegicus)YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO:3) Mouse (Mus musculus)YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO:4) PigYPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO:5) Guinea pigYPSKPEAPGSDASPEELARYYASLRHYLNLVTRQRY (SEQ ID NO:6) FrogYPPKPENPGEDASPEEMTKYLTALRHYINLVTRQRY (SEQ ID NO:7) RajaYPPKPENPGDDAAPEELAKYYSALRHYINLITRQRY (SEQ ID NO:8) DogfishYPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY (SEQ ID NO:9) LampetraFPPKPDNPGDNASPEQMARYKAAVRHYINLITRQRY (SEQ ID NO:10) PetromyzonMPPKPDNPSPDASPEELSKYMLAVRNYINLITRQRY (SEQ ID NO:11) Dog (Canisfamiliaris) YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO:12) Rhesusmonkey (Macaca mulatta) YPIKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ IDNO:13) Pipid frog (Xenopus tropicalis)YPTKPENPGNDASPEEMAKYLTALRHYINLVTRQRY (SEQ ID NO:14) Atlantic salmon(Salmo salar) YPPKPENPGEDAPPEELAKYYTALRHYINLITRQRY (SEQ ID NO:15) Cattle(bos taurus) YPAKPQAPGEHASPDELNRYYTSLRHYLNLVTRQRF (SEQ ID NO:16)

Peripheral Administration: Administration outside of the central nervoussystem. Peripheral administration does not include direct administrationto the brain. Peripheral administration includes, but is not limited tointravascular, intramuscular, subcutaneous, inhalation, oral, rectal,transdermal or intra-nasal administration.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein encompass any amino acid sequence and includemodified sequences such as glycoproteins. The term “polypeptide” isspecifically covers naturally occurring proteins, as well as those whichare recombinantly or synthetically produced. The term “polypeptidefragment” refers to a portion of a polypeptide, for example a fragmentwhich exhibits at least one useful sequence in binding a receptor. Theterm “functional fragments of a polypeptide” refers to all fragments ofa polypeptide that retain an activity of the polypeptide. Biologicallyfunctional peptides can also include fusion proteins, in which thepeptide of interest has been fused to another peptide that does notdecrease its desired activity.

Subcutaneous administration: Subcutaneous administration isadministration of a substance to the subcutaneous layer of fat which isfound between the dermis of the skin and the underlying tissue.Subcutaneous administration may be by an injection using a hypodermicneedle fitted, for example, to a syringe or a “pen” type injectiondevice. Other administration methods may be used for examplemicroneedles. Injection with a hypodermic needle typically involves adegree of pain on behalf of the recipient. Such pain may be masked byuse of a local anaesthetic or analgesic. However, the usual method usedto reduce the perceived pain of injections is to merely distract thesubject immediately prior to and during the injection. Pain may beminimised by using a relatively small gauge hypodermic needle, byinjecting a relatively small volume of substance and by avoidingexcessively acidic or alkali compositions which may cause the subject toexperience a “stinging” sensation at the injection site. Compositionshaving a pH of between pH4 and pH10 are usually regarded as tolerablycomfortable.

Therapeutically effective amount: A dose sufficient to preventadvancement, or to cause regression of a disorder, or which is capableof relieving a sign or symptom of a disorder, or which is capable ofachieving a desired result. In several embodiments, a therapeuticallyeffective amount of a compound of the invention is an amount sufficientto inhibit or halt weight gain, or an amount sufficient to decreaseappetite, or an amount sufficient to reduce caloric intake or foodintake or increase energy expenditure.

DETAILED DESCRIPTION

According to a first aspect of the invention there is provided ananalogue of PYY which differs from the sequence of native human PYY(Tyr1 Pro2 Ile3 Lys4 Pro5 Glu6 Ala7 Pro8 Gly9 Glu10 Asp11 Ala12 Ser13Pro14 Glu15 Glu16 Leu17 Asn18 Arg19 Tyr20 Tyr21 Ala22 Ser23 Leu24 Arg25His26 Tyr27 Leu28 Asn29 Leu30 Val31 Thr32 Arg33 Gln34 Arg35 Tyr36; SEQID NO:1) in the following respect:

-   Ser23 is substituted with Ala23, Glu23, Lys23, Gln23 or AIB23    (preferably Ala23) and which may further differ from the sequence of    native human PYY in one of more of the following further respects:-   Tyr1 is absent,-   one or more of residues 2 to 9, 13 to 16, 20, 21, 24, 25 or 26    (preferably, residues 11, 18, 19, 23, 27 and 30 or at least residues    11, 18, 19, 23, 27 and 30, or at least 4 or 5 of residues selected    from 11, 18, 19, 23, 27 and 30) are subject to a conservative    substitution,-   Glu10 is substituted with Ala10, LyslO or Gln10-   Asp11 is substituted with Gly11-   Ala12 is substituted with AIB12-   Leu17 is substituted with Ile17 or AIB17-   Asn18 is substituted with AIB18, Ala18 or Leu18 (preferably Leu18)-   Arg19 is substituted with His19 or Lys19 (preferably His19)-   Ala22 is substituted with Val22 or Ile22-   Tyr27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31

-   and wherein the C-terminal residue optionally terminates in a    primary amide (-C(O)NH₂) group in place of a carboxylic acid group    (-CO₂H);-   or a derivative of the compound; or a salt of the compound or the    derivative.

The amino acid sequence of formula (I) above is shown with theN-terminus to the top left and the C-terminus to the bottom right.Unless indicated otherwise, the amino acid residues in the sequence offormula (I) are L-amino acids.

In one preferred embodiment, Tyr1 is absent. The removal of Tyr1increases the selectivity of a PYY analogue for the Y2 receptor over theY1 receptor, agonism of the Y2 receptor being responsible for theinhibition of feeding.

In one preferred embodiment residues 2 to 9 are unsubstituted.

In another preferred embodiment residues 2 to 9 are unsubstituted andTyr1 is absent.

In another embodiment Tyr1 is absent, residues 2 to 9 are unsubstitutedand Glu10 is substituted with LyslO.

In another embodiment Tyr1 is absent, residues 2 to 9 are unsubstitutedand Glu10 is substituted with Gln10.

In another especially preferred embodiment Tyr1 is absent and residues 2to 10 are unsubstituted.

According to one embodiment

-   Tyr1 is absent-   residues 2 to 9, 13 to 16, 20, 21, 24, 25, 26, 28, 29, 32, 33, 34    and 35 are unsubstituted.

According to one especially preferred embodiment:

-   Tyr1 is absent-   residues 2 to 10, 12 to 17, 20 to 22, 24 to 26, 28, 29 and 31 to 36    are unsubstituted. Optionally, residue 14 may also be unsubstituted.

According to one embodiment:

-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is substituted with AIB18, Ala18 or Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted-   Ser23 is substituted with Glu23, Ala23, AIB 23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31.

According to one embodiment:

-   Tyr1 is absent-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is substituted with AIB18, Ala18 or Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted-   Ser23 is substituted with Glu23, Ala23, AIB 23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31.

According to one embodiment:

-   Tyr1 is absent-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residues 2 to 9, 12 to 17, 20, 21, 24, 25, 26, 28, 29, 32, 33, 34    and 35 are unsubstituted-   Asn18 is substituted with AIB18, Ala18 or Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted-   Ser23 is substituted with Glu23, Ala23, AIB 23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31.

According to one embodiment:

-   Tyr1 is absent-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residues 2 to 9, 12 to 17, 20, 21, 24, 25, 26, 28, 29, 32, 33, 34    and 35 are unsubstituted-   Asn18 is substituted with AIB18, Ala18 or Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted-   Ser23 is substituted with Glu23, Ala23, AIB 23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31-   the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H).

According to one embodiment:

-   Glu10 is unsubstituted-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is unsubstituted-   Arg19 is His19-   Ala22 is unsubstituted-   Ser23 is substituted with Ala23-   Tyr27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31-   the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H).

According to one embodiment:

-   Tyr1 is absent-   Glu10 is unsubstituted-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is unsubstituted-   Arg19 is His19-   Ala22 is unsubstituted-   Ser23 is substituted with Ala23-   Tyr27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31-   the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H); optionally all of    residues 2 to 9, 12 to 17, 20, 21, 24, 25, 26, 28, 29, 32, 33, 34    and 35 are unsubstituted.

In some embodiments Asp11 is substituted with Gly11.

In some embodiments Asn18 is substituted with AIB18, Ala18 or Leu18,preferably Leu18.

According to one embodiment:

-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is substituted with Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted-   Ser23 is substituted with Glu23, Ala23, AIB 23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31.

According to one embodiment:

-   Tyr1 is absent-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is substituted with Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted Ser23 is substituted with Glu23, Ala23, AIB    23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31.

According to one embodiment:

-   Tyr1 is absent-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residues 2 to 9, 12 to 17, 20, 21, 24, 25, 26, 28, 29, 32, 33, 34    and 35 are unsubstituted-   Asn18 is substituted with Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted-   Ser23 is substituted with Glu23, Ala23, AIB 23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31.

According to one embodiment:

-   Tyr1 is absent-   Glu10 is substituted with LyslO or Gln10-   Asp11 is substituted with Gly11-   Residues 2 to 9, 12 to 17, 20, 21, 24, 25, 26, 28, 29, 32, 33, 34    and 35 are unsubstituted-   Asn18 is substituted with Leu18-   Arg19 is substituted with His19-   Ala22 is unsubstituted-   Ser23 is substituted with Glu23, Ala23, AIB 23 or Gln23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31-   the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H).

According to one embodiment:

-   Glu10 is unsubstituted-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is substituted with Leu18-   Arg19 is His19-   Ala22 is unsubstituted-   Ser23 is substituted with Ala23-   Tyr27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31-   the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H).

According to one embodiment:

-   Tyr1 is absent-   Glu10 is unsubstituted-   Asp11 is substituted with Gly11-   Residue 12 is unsubstituted-   Leu17 is unsubstituted-   Asn18 is substituted with Leu18-   Arg19 is His19-   Ala22 is unsubstituted-   Ser23 is substituted with Ala23-   Tyr27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31-   the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H)-   optionally all of residues 2 to 9, 12 to 17, 20, 21, 24, 25, 26, 28,    29, 32, 33, 34 and 35 are unsubstituted.

According to one embodiment:

-   Tyr1 is absent-   Residues 2 to 10, 12 to 17, 20, 21, 22, 24, 25, 26, 28, 29, 31, 32,    33, 34, 35 and 36 are unsubstituted-   Asp11 is substituted with Gly11-   Asn18 is substituted with Leu18-   Arg19 is substituted with His19-   Ser23 is substituted with Ala23-   Tyr 27 is substituted with Phe27-   Leu30 is substituted with His30-   Val31 is substituted with Leu31-   the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H).

According to one preferred embodiment at least 9 of the following 10criteria apply:

-   1 - Tyr 1 is absent-   2 - residues 2 to 10, 12 to 17, 20, 21, 22, 24, 25, 26, 28, 29, 31,    32, 33, 34, 35 and 36 are unsubstituted-   3 - Asp11 is substituted with Gly11-   4 - Asn18 is substituted with Leu18-   5 - Arg19 is substituted with His19-   6 - Ser23 is substituted with Ala23-   7 - Tyr27 is substituted with Phe27-   8 - Leu30 is substituted with His30-   9 - Val31 is unsubstituted-   10 - the C-terminal residue terminates in a primary amide (—C(O)NH₂)    group in place of a carboxylic acid group (—CO₂H).

The invention contemplates embodiments above, wherein at least criteria1 and 2 and at least 7 other criteria apply, or where criteria 1, 2 and10 and at least 6 other criteria apply, or where all criteria 1 to 10apply.

According to some embodiments Leu30 is substituted with His30.

According to some embodiments Val31 is unsubstituted.

According to certain embodiments, the naturally occurring Pro residuesat positions 2, 5, 8 and 14 are retained. It has been found that theseresidues form hydrogen bonds which are important in stabilizing thetertiary structure of the molecule. Accordingly, according to certainpreferred embodiments, there is provided an analogue of PYY whichdiffers from the sequence of native human PYY in the following respects:

- Ser23 is substituted with Ala23, Glu23, Lys23, Gln23 or AIB23(preferably Ala23)

Such a compound may further differ from the sequence of native human PYYin one or more of the following respects:

-   — Tyr is absent-   One or more residues 3, 4, 6, 7, 9, 13, 15, 16, 20, 21, 24, 25 or 26    are subject to a conservative substitution,-   Glu10 is substituted with Ala10, LyslO or Gln10-   Asp11 is substituted with Gly11-   Ala12 is substituted with AIB12-   Leu17 is substituted with Ile17 or AIB17-   Asn18 is substituted with AIB18, Ala18 or Leu18 (preferably Leu18)-   Arg19 is substituted with His19 or Lys19 (preferably His19)-   Ala22 is substituted with Val22 or Ile22-   Tyr27 is substituted with Phe27-   -Leu30 is substituted with His30-   Val31 is substituted with Leu31

-   and wherein the C-terminal residue optionally terminates in a    primary amide (—C(O)NH₂) group in place of a carboxylic acid group    (—CO₂H);-   or a derivative of the compound; or a salt of the compound or the    derivative.

According to all embodiments it is preferred that the C-terminal residueterminates in a primary amide group (—C(O)NH₂) in place of a carboxylicacid group (—CO₂H). The absence of the amide group results in asignificant decrease in agonism of the Y2 receptor.

According to all embodiments Ser23 is substituted for an alternativeamino acid residue. Although a substitution at position 23 haspreviously been disclosed as a tolerated change from the nativesequence, previous PYY analogues which contained position 23substitutions (for example Y242 disclosed in the applicant’s previouspatent applications) did not exhibit the same advantages as peptides ofthe present invention as shown in the comparative studies presentedherein.

According to some embodiments Ser23 is substituted with Glu23, Ala23,AIB23 or Gln23.

Preferred specific sequences include the sequences disclosed herein andin particular those identified as Y1319, Y1371, Y1372, Y1419, Y1421,Y1518, Y1528, Y1558, Y1568, Y1572, Y1579, Y1553 or Y1581 (and especiallyY1419). The invention encompasses, as preferred sequences, sequencescorresponding to those of Y1319, Y1371, Y1372, Y1419, Y1421, Y1518,Y1528, Y1558, Y1568, Y1572, Y1579, Y1553 or Y1581 (and especially Y1419)to which 1 or 2 substitutions (for example conservative substitutionshave been made) and which retain the activity (for example at least 20,50 or 70% of the activity in one of the assays disclosed herein) of thesequence on which it is based.

According to a further aspect of the invention there is provided apharmaceutical composition comprising an analogue of PYY according tothe invention together with a pharmaceutically acceptable carrier andoptionally a further therapeutic agent.

The second therapeutic agent may be an anti-obesity agent, an appetitesuppressant or an anti-diabetic agent. Preferably the therapeutic agentis an analogue of GLP-1 or an analogue of glucagon or a derivative ofeither thereof.

According to certain embodiments discussed further below the compositioncomprises Zn²⁺ ions.

According to another aspect of the invention there is provided ananalogue of PYY according to the invention or a pharmaceuticalcomposition according to the invention for use as a medicament.

According to certain embodiments the medicament is for the prevention ortreatment of a disorder of energy metabolism, such as diabetes and/orobesity or for preventing loss of pancreatic islet function or forrecovering pancreatic islet function.

According to certain embodiments the medicament is for use in preventingdiabetes or obesity in a subject.

According to a further aspect of the invention there is provided amethod of treating or preventing a disease or disorder or othernon-desired physiological state in a subject comprising administering atherapeutically acceptable/ effective amount of an analogue of PYYaccording to the invention, or a pharmaceutical composition, accordingto the invention, to the subject.

According to a further aspect of the invention there is provided amethod of preventing or treating a disorder of energy metabolism such asdiabetes and/or obesity, preventing loss of pancreatic islet functionand/or recovering pancreatic islet function, reducing appetite, reducingfood intake, and/or reducing calorie intake in a subject, comprisingadministering a therapeutically effective amount of an analogue of PYYaccording to the invention, or a pharmaceutical composition as definedin claims according to the invention to the subject.

According to a further aspect of the invention there is provided amethod of preventing or treating diabetes and/or obesity in a subject,comprising administering a therapeutically effective amount of ananalogue of PYY according to the invention, or a pharmaceuticalcomposition according to the invention, to the subject.

In respect of all aspects of the invention, the subject may beoverweight, obese and/or diabetic. Alternatively the subject may be of ahealthy weight.

The PYY analogue or pharmaceutical composition according to theinvention is administered parentally. According it may be provided in aformat suitable for such administration. For example it may be providedin an injection device.

Preferably the PYY analogue or pharmaceutical composition isadministered subcutaneously and may be provided in a format suitable forsuch administration.

According to a further aspect of the invention there is provided amethod of causing weight loss or preventing weight gain in a subject forcosmetic purposes, comprising administering an effective amount of ananalogue of PYY according to the invention, to the subject. The subjectin such cases may be obese, overweight or according to some preferredembodiments of healthy weight.

Peptide hormone analogues of the invention may be produced byrecombinant methods well known in the art or alternatively they may beproduced by synthetic methods, again well known in the art.

PYY analogues according to the present invention preferably have a moresustained effect on food intake reduction or have a stronger effect onfood intake reduction than human PYY. Preferably they have an effect onfood intake reduction which is at least as strong as native human PYYbut which is more sustained. Increased duration of appetite suppressioncan be particularly important to avoid the effect known as “escape”. Ashort duration of appetite suppressant may reduce appetite or the timecovered by one meal and in that meal the subject typically eats lessfood. If, however, the appetite suppressant is then metabolized orotherwise removed from circulation as a subject then by the time thenext meal the subject can regain its “normal” appetite. In view of thesubject having eaten a small meal at the previous mealtime, the subjectmay in fact have an increased appetite at the time of the second meal.If the subject satisfies that appetite it is possible for the foodintake over the two meals in total to be no lower than the food intakewould have been without the appetite suppressant. That is to say, thesubject may have “escaped” from the effects of the appetite suppressant.“Escape” can be reduced by using additional doses of appetitesuppressant or by using an appetite suppressant with a longer durationof action. If the subject has a reduced appetite for longer, then thedegree to which it can make up the deficit from one meal in the nextmeal is reduced and as there is a practical limit to total capacity in aparticular single meal.

Preferably the PYY analogues of the invention are selective for the Y2receptor. That is say, they bind with a higher affinity to Y2 comparedwith other receptors such as Y1, Y3, Y4, Y5 and Y6. Those receptors arerecognized based on binding affinity, pharmacology and sequence. Most,if not all, of the receptors are G protein coupled receptors. The Y1receptor is generally considered to be postsynaptic and mediates many ofthe known actions of neuropeptide Y in the periphery. Originally, thisreceptor was described as having poor affinity for C-terminal fragmentsof neuropeptide Y, such as the 13-36 fragment, but interacts with thefull length neuropeptide Y and peptide YY with equal affinity (see PCTpublication WO 93/09227).

Pharmacologically, the Y2 receptor is distinguished from Y1 byexhibiting affinity for C-terminal fragments of neuropeptide Y. The Y2receptor is most often differentiated by the affinity of neuropeptideY(13-36), although the 3-36 fragment of neuropeptide Y and peptide YYprovides improved affinity and selectivity (see Dumont et al., Societyfor Neuroscience Abstracts 19:726, 1993). Signal transmission throughboth the Y1 and the Y2 receptors are coupled to the inhibition ofadenylate cyclase. Binding to the Y2 receptor was also found to reducethe intracellular levels of calcium in the synapse by selectiveinhibition of N-type calcium channels. In addition, the Y2 receptor,like the Y1 receptors, exhibits differential coupling to secondmessengers (see U.S. Pat. No. 6,355,478). Y2 receptors are found in avariety of brain regions, including the hippocampus, substantianigra-lateralis, thalamus, hypothalamus, and brainstem. The human,murine, monkey and rat Y2 receptors have been cloned (e.g., see U.S.Pat. No. 6,420,352 and U.S. Pat. No. 6,355,478). Preferably the PYYanalogue of the invention binds to Y2 with an affinity that is at leasttwo-fold, at least five-fold, or at least tenfold greater than thebinding affinity for Y1, Y3, Y4, Y5 and/or Y6.

An analogue of PYY according to the invention preferably has low or nonet ionic charge in solution (i.e., in a solution approximating tophysiological conditions, such as, for example, those found in thetissue fluid or plasma, such as at pH 7.4), e.g. preferably the PYYanalogue of the invention has a net ionic charge at pH 7.4 of +1, 0, or-1. For example, a PYY analogue having two acidic groups and two basicgroups will have a net ionic charge of 0. An acidic group which has apKa in water of less than 6.4 is considered as contributing an ioniccharge of -1 at pH 7.4. A basic group which has a pKa of more than 8.4is considered as contributing an ionic charge of +1 at pH 7.4. It ishypothesised that low net ionic charge, and particularly an absence ofnet ionic charge under in vivo conditions, limits in vivo solubility ofthe PYY analogue, and that this contributes to a slower absorption aftersubcutaneous administration of a high concentration peptide and thusprolonged presence in the circulation.

PYY analogues according to the invention contain histidine at position26, as is the case for native PYY. PYY analogues according to theinvention preferably also contain His at position 19 and/or 30.According to one preferred aspect of the invention, analogues of PYYaccording to the invention further contain at least one of the aminoacids corresponding to positions 19 and 30 in the native PYY moleculesubstituted for histidine. More preferably, the amino acid at position30 of the PYY analogue is histidine. In one particularly preferredembodiment, the PYY analogue of the invention contains 3 histidineresidues, at positions 19, 26 and 30.

By way of further explanation, histidine is a unique amino acid in beingnot charged at pH 7.4 (i.e. under physiological conditions in thecirculation or subcutaneously following subcutaneous administration).However, it is fully charged at pH 5 (or lower) since the pI of the NHside chain of histidine is about 6.0. According to certain preferredembodiments an analogue of PYY according to the invention has low or nooverall charge at physiological pH (pH 7.4) and is preferably formulatedas part of a composition having a pH of about pH5 (for example from pH4.5 to pH 6.0 - a lower pH than approximately pH 4 or 5 may beundesirable for an injectable composition because it is likely toincrease pain at the injection site) so as to exhibit histidineionisation and preferably an overall net change at such a lower pH. Anincrease in the number of charged residues increases the solubility ofan injectable composition in the vial and therefore allows a smallvolume injection of a relatively concentrated peptide solution to begiven. However, subsequent to subcutaneous injection the analogue isexposed to physiological pH at which the number of ionised residues andespecially the number of ionised histidine residues falls and thereforesolubility decreases. This causes the peptide to precipitatesubcutaneously.

The presence of His residues enhances this effect.

According to certain preferred embodiments, PYY analogues according tothe invention have a combination of the following preferred features:

-   1) A peptide sequence which at pH 7.4 has no or low net ionic charge    (e.g. +1, 0, or -1) and may have relatively few charged groups and    hydrophilic groups overall to decrease intrinsic solubility.-   2) The presence of a number of histidines which produce a net    positive charge and good solubility at pH 5 for storage before    administration and to allow a low viscosity administration solution    (at pH 5).-   3) Suitability for subcutaneous administration of a low volume and    high concentration, exceeding the solubility constant at pH 7.4 but    not at pH 5.

In addition to histidine being a particularly advantageous amino acidresidue for causing this differential pH-dependent solubility effect,the differential solubility of peptides containing histidine residues isgreatly enhanced if formulated together with zinc ions. This is becausezinc ions will bind to uncharged histidine residues in aqueous solution.It is believed that zinc ions are able to bind simultaneously to up to 4uncharged histidines. This allows zinc to co-ordinate with histidineresidues in several individual peptide molecules and thereby weaklycross-link the peptide molecule to other similar peptide moleculesleading to a fall in solubility. However, zinc ions do not bind tocharged histidine. Therefore, histidine containing peptides in acomposition containing zinc ions will be cross-linked by weak ionicbonds at pH 7.4 but not at pH 5.0. The presence of His residues bound tozinc ions therefore enhances precipitation of the peptide aftersubcutaneous injection but does not affect solubility in the vial orsyringe before administration. This means that a peptide having anoverall pI of approximately 7 will have no charged residues atapproximately neutral pH and a peptide comprising histidine residues ina formulation including zinc ions is advantageously soluble in the vialor syringe but precipitates subcutaneously following administration. Soa pH 7 neutral peptide with histidines in a formulation including zincions is advantageously soluble in the vial and syringe but precipitatessubcutaneously following administration. Furthermore, zinc-enhancedprecipitation is gradually reversible because the concentration of zincions following injection will fall as zinc ions are gradually washed outof the injection site. Therefore there is observed a delay insubcutaneous absorption with much better pharmacokinetics but no loss ofbio-availability. The rate of absorption for a givenhistidine-containing neutral peptide can be controlled by the amount ofzinc added.

Introduction of at least one additional histidine residue preferablyresults in the PYY analogues of the invention having at least oneoccurrence of two histidine residues separated from each other by 1 to 3intervening amino acid residues (a pair of histidine residues). Such aspacing appears to be optimum for a single zinc ion to form in aqueoussolution associations with both histidine residues in a pair. In oneadvantageous embodiment of the invention the amino acid residues atpositions 26 and 30 are each histidine residues.

Preferably an analogue according to the invention has an overall pI ofbetween 6.5 and 8.5. This means that at physiological pH (e.g. pH 7.4)the analogue has no low or no significant overall charge (e.g. asmentioned above, preferably the PYY analogue of the invention has a netionic charge at pH 7.4 of +1, 0, or -1). The overall pI of a moleculemay be calculated using techniques well known to a person skilled in theart or alternatively may be determined experimentally by usingisoelectric focusing.

In order to take full advantage of this effect the inventors have foundthat the following combination of features are particularly preferred.

-   1) Peptide sequence which at pH 7.4 has low or no net ionic charge    (e.g. +1, 0, or -1).-   2) Presence of three histidines which produce a net positive charge    and good solubility at pH 5 for storage before administration.-   3) High solubility at pH 5 which allows for subcutaneous    administration of a therapeutic dose of the PYY analogue in a low    volume of aqueous medium, and low solubility at pH 7.4.-   4) The presence of zinc ions which produce cross-linking of    uncharged histidine residues at pH 7.4 and adjacent molecules but    which do not cross-link charge histidine at pre-administration pH or    approximately pH 5.

It is found that analogues having a His residue at positions 19 and 30may be especially advantageous and therefore to be preferred. TheInventors have found that, despite the last six amino acids of thesequence being thought to be important for the activity of the compound,the native Leu30 can be substituted for His30 without loss of activity.

Accordingly, according to certain preferred embodiments, there isprovided an analogue of PYY which differs from the sequence of nativePYY in the following respects:

-   Ser23 is substituted with Ala23, Glu23, Lys23, Gln23 for AIB23    (preferably Ala23)-   Arg19 is substituted with His19-   Leu30 is substituted with His30

Such a compound may further differ from the sequence of native human PYYin one or more of the following respects:

-   Tyr is absent-   One or more of residues 2, 4, 6, 7, 9, 13, 15, 16, 20, 21, 24, 25 or    26 are subject to a conservative substitution-   Glu10 is substituted with Ala10, LyslO or Gln10-   Asp11 is substituted with Gly11-   Ala12 is substituted with AIB12-   Leu17 is substituted with Ile17 or AIB17-   Asn18 is substituted with AIB18, Ala18 or Leu18 (preferably Leu18)-   Ala22 is substituted with Val22 or Ile22-   Tyr27 is substituted with Phe27-   Val31 is substituted with Leu31

and wherein the C-terminal residue optionally terminates in a primaryamide (—C(O)NH₂) group in place or a carboxylic acid group (—CO₂H); or aderivative of the compound; or a salt of the compound or the derivative.

Preferably residues 2, 4, 6, 7, 9, 13, 15, 16, 20, 21, 24, 25 and 26 arenot substituted.

Derivatives

A PYY analogue of the invention may be a derivative as a consequence ofbeing modified by well-known processes including amidation,glycosylation, carbamylation, acylation, for example acetylation,sulfation, phosphorylation, cyclization, lipidization, pegylation andfusion to another peptide or protein to form a fusion protein. A PYYanalogue of the invention may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

A PYY analogue of the invention may be a fusion protein, whereby theanalogue is fused to another protein or polypeptide (the fusion partner)using recombinant methods known in the art. Alternatively, such a fusionprotein may be synthetically synthesized by any known method. Anysuitable peptide or protein can be used as the fusion partner (e.g.,serum albumin, carbonic anhydrase, glutathione-S-transferase orthioredoxin, etc.). Preferred fusion partners will not have an adversebiological activity in vivo. Such fusion proteins may be made by linkingthe carboxy-terminus of the fusion partner to the amino-terminus of thePYY analogue or vice versa. Optionally, a cleavable linker may be usedto link the PYY analogue to the fusion partner. A resulting cleavablefusion protein may be cleaved in vivo such that an active form of acompound of the invention is released. Examples of such cleavablelinkers include, but are not limited to, the linkers D—D—D—D—Y, G—P—R,A-G-G and H—P—F—H—L, which can be cleaved by enterokinase, thrombin,ubiquitin cleaving enzyme and renin, respectively. See, e.g., U.S. Pat.No. 6,410,707, the contents of which are incorporated herein byreference. According to certain embodiments of all aspects of theinvention, the PYY analogue is not a fusion protein.

A PYY analogue of the invention may be a physiologically functionalderivative. The term “physiologically functional derivative” is usedherein to denote a chemical derivative of a PYY analogue of theinvention having the same physiological function as the correspondingunmodified PYY analogue of the invention. For example, a physiologicallyfunctionally derivative may be convertible in the body to a PYY analogueof the invention. According to the present invention, examples ofphysiologically functional derivatives include esters, amides, andcarbamates; preferably esters and amides.

Pharmaceutically acceptable esters and amides of the compounds of theinvention may comprise a C₁₋₂₀ alkyl-, C₂₋₂₀ alkenyl-, C₅₋₁₀ aryl-,C₅₋₁₀ ar-C₁₋₂₀ alkyl-, or amino acid-ester or -amide attached at anappropriate site, for example at an acid group. Examples of suitablemoieties are hydrophobic substituents with 4 to 26 carbon atoms,preferably 5 to 19 carbon atoms. Suitable lipid groups include, but arenot limited to, the following: lauroyl (C₁₂H₂₃), palmityl (C₁₅H₃₁),oleyl (C₁₅H₂₉), stearyl (C₁₇H₃₅), cholate; and deoxycholate.

Methods for lipidization of sulfhydryl-containing compounds with fattyacid derivatives are disclosed in U.S. Pat. No. 5,936,092; U.S. Pat. No.6,093,692; and U.S. Pat. No. 6,225,445. Fatty acid derivatives of acompound of the invention comprising a compound of the invention linkedto fatty acid via a disulfide linkage may be used for delivery of acompound of the invention to neuronal cells and tissues. Lipidisationmarkedly increases the absorption of the compounds relative to the rateof absorption of the corresponding unlipidised compounds, as well asprolonging blood and tissue retention of the compounds. Moreover, thedisulfide linkage in lipidised derivative is relatively labile in thecells and thus facilitates intracellular release of the molecule fromthe fatty acid moieties. Suitable lipid-containing moieties arehydrophobic substituents with 4 to 26 carbon atoms, preferably 5 to 19carbon atoms. Suitable lipid groups include, but are not limited to, thefollowing: palmityl (C₁₅H₃₁), oleyl (C₁₅H₂₉), stearyl (C₁₇H₃₅), cholate;and deoxycholate.

Cyclization methods include cyclization through the formation of adisulfide bridge and head-to-tail cyclization using a cyclization resin.Cyclized peptides may have enhanced stability, including increasedresistance to enzymatic degradation, as a result of their conformationalconstraints. Cyclization may in particular be expedient where theuncyclized peptide includes an N-terminal cysteine group. Suitablecyclized peptides include monomeric and dimeric head-to-tail cyclizedstructures. Cyclized peptides may include one or more additionalresidues, especially an additional cysteine incorporated for the purposeof formation of a disulfide bond or a side chain incorporated for thepurpose of resin-based cyclization.

A PYY analogue of the invention may be a pegylated structure of a PYYanalogue. Pegylated compounds of the invention may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337).

Chemical moieties for derivitisation of a compound of the invention mayalso be selected from water soluble polymers such as polyethyleneglycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Apolymer moiety for derivatisation of a compound of the invention may beof any molecular weight, and may be branched or unbranched. For ease inhandling and manufacturing, the preferred molecular weight of apolyethylene glycol for derivatisation of a compound of the invention isfrom about 1 kDa to about 100 kDa, the term “about” indicating that inpreparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight. Polymers of other molecularweights may be used, depending on the desired therapeutic profile, forexample the duration of sustained release desired, the effects, if anyon biological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to atherapeutic protein or analog. For example, the polyethylene glycol mayhave an average molecular weight of about 200, 500, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500,13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000,17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000,40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,90,000, 95,000, or 100,000 kDa.

In one embodiment, the PYY analogue of the invention is not aderivative.

Salts of PYY analogues of the invention that are suitable for use in amedicament are those wherein a counterion is pharmaceuticallyacceptable. However, salts having non-pharmaceutically acceptablecounterions are within the scope of the present invention, for example,for use as intermediates in the preparation of PYY analogues of theinvention and their pharmaceutically acceptable salts and/or derivativesthereof.

Suitable salts according to the invention include those formed withorganic or inorganic acids or bases. Pharmaceutically acceptable acidaddition salts include those formed with hydrochloric, hydrobromic,sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic,pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic,glycollic, lactic, salicylic, oxaloacetic, methanesulfonic,ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic, benzenesulfonic, and isethionic acids.

Pharmaceutically acceptable salts with bases include ammonium salts,alkali metal salts, for example potassium and sodium salts, alkalineearth metal salts, for example calcium and magnesium salts, and saltswith organic bases, for example dicyclohexylamine andN-methyl-D-glucomine.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Thesecomplexes are known as “solvates”. For example, a complex with water isknown as a “hydrate”. Solvates, such as hydrates, exist when the drugsubstance incorporates solvent, such as water, in the crystal lattice ineither stoichiometric or non-stoichiometric amounts. Drug substances areroutinely screened for the existence of hydrates since these may beencountered at any stage of the drug manufacturing process or uponstorage of the drug substance or dosage form. Solvates are described inS. Byrn et al, Pharmaceutical Research 12(7), 1995, 954-954, andWater-Insoluble Drug Formulation, 2^(nd) ed. R. Liu, CRC Press, page553, which are incorporated herein by reference. Accordingly, it will beunderstood by the skilled person that PYY analogues of the invention, aswell as derivatives and/or salts thereof may therefore be present in theform of solvates. Solvates of PYY analogues of the invention which aresuitable for use in medicine are those wherein the associated solvent ispharmaceutically acceptable. For example, a hydrate is an example of apharmaceutically acceptable solvate.

Conditions

The invention also provides an analogue of PYY according to theinvention, or a pharmaceutical composition comprising the analogue ofPYY, for use as a medicament. The PYY analogue and pharmaceuticalcomposition find use in the treatment and/or prevention of conditionssuch as diabetes and obesity. The PYY analogue, and pharmaceuticalcomposition comprising the PYY analogue, also find use in reducingappetite in a subject, reducing food intake in a subject, and/orreducing calorie intake in a subject.

The invention also provides the use of an analogue of PYY according tothe invention for the manufacture of a medicament for the prevention ortreatment of diabetes and/or obesity. The invention also provides theuse of an analogue of PYY according to the invention for the manufactureof a medicament for reducing appetite in a subject, reducing food intakein a subject, and/or reducing calorie intake in a subject.

The invention also provides a method of treating or preventing a diseaseor disorder or other non-desired physiological state in a subject,comprising administering a therapeutically effective amount of ananalogue of PYY according to the invention, or a pharmaceuticalcomposition comprising the PYY analogue, to the subject.

The invention also provides a method of preventing or treating diabetesand/or obesity, reducing appetite, reducing food intake, and/or reducingcalorie intake in a subject, comprising administering a therapeuticallyeffective amount of an analogue of PYY according to the invention, or apharmaceutical composition comprising the PYY analogue, to the subject.

In one embodiment, the PYY analogue or pharmaceutical composition isadministered parentally. In one embodiment, the PYY analogue orpharmaceutical composition is administered subcutaneously. In oneembodiment, the PYY analogue or pharmaceutical composition isadministered intravenously, intramuscularly, intranasally, transdermallyor sublingually.

The subject to whom the PYY analogue according to the invention, orpharmaceutical composition comprising the PYY analogue, is administeredmay be overweight, for example they may be obese. Alternatively, or inaddition, the subject may be diabetic, for example having insulinresistance or glucose intolerance, or both. The subject may havediabetes mellitus, for example, the subject may have Type II diabetes.The subject may be overweight, for example, obese and have diabetesmellitus, for example, Type II diabetes. Alternatively, the subject mayhave Type I diabetes.

The PYY analogues of the invention are thought to protect islet ofLangerhans cells, in particular beta cells, allowing them to retaintheir normal physiological function, for example the ability to secreteinsulin in response to appropriate stimuli, when challenged by toxins(e.g. streptozotocin), pathogens or by an autoimmune response. The PYYanalogues of the invention are also thought to be effective inrecovering or rescuing pancreatic islet function, and, in particular,beta cell function, following deterioration of physiological functionfollowing exposure to a toxin, pathogen or an autoimmune response.Recovery of function may be to at least 10, 20, 30, 40, 50, 60, 70, 80,90 or 100 % of the function exhibited prior to deterioration.Accordingly, the invention also provides a PYY analogue of theinvention, or a pharmaceutical composition comprising the PYY analogue,for use in preventing loss of pancreatic islet function (for examplebeta cell function) and/or recovering pancreatic islet function (forexample beta cell function). The invention further provides the use of aPYY analogue of the invention for the manufacture of a medicament forpreventing loss of pancreatic islet function (for example beta cellfunction) and/or for recovering pancreatic islet function (for examplebeta cell function). The invention further provides a method ofpreventing loss of pancreatic islet function (for example beta cellfunction) and/or recovering pancreatic islet function (for example betacell function) in a subject comprising administering to the subject aneffective amount of a PYY analogue of the invention, or a pharmaceuticalcomposition comprising the PYY analogue, to the subject.

The pancreatic islet-protecting properties of the PYY analogues of theinvention render them useful for administration in combination withfurther therapeutic agents which have as a side-effect islet toxicity.An example of such a therapeutic agent is steptozocin. Accordingly, theinvention also provides a PYY analogue according to the invention incombination with a further therapeutic agent which has islet toxicity asa side-effect. The invention also provides a pharmaceutical compositioncomprising a PYY analogue according to the invention and a furthertherapeutic agent which has islet toxicity as a side-effect, togetherwith a pharmaceutically acceptable carrier.

In addition, or alternatively, the subject may have, or may be at riskof having, a disorder in which obesity or being overweight is a riskfactor. Such disorders include, but are not limited to, cardiovasculardisease, for example hypertension, atherosclerosis, congestive heartfailure, and dyslipidemia; stroke; gallbladder disease; osteoarthritis;sleep apnea; reproductive disorders for example, polycystic ovariansyndrome; cancers, for example breast, prostate, colon, endometrial,kidney, and esophagus cancer; varicose veins; acnthosis nigricans;eczema; exercise intolerance; insulin resistance; hypertensionhypercholesterolemia; cholithiasis; osteoarthritis; orthopedic injury;insulin resistance, for example, type-2 diabetes and syndrome X; andthromboembolic disease (see Kopelman, Nature 404:635-43; Rissanen etal., British Med. J. 301, 835, 1990).

Other disorders associated with obesity include depression, anxiety,panic attacks, migraine headaches, PMS, chronic pain states,fibromyalgia, insomnia, impulsivity, obsessive compulsive disorder, andmyoclonus. Furthermore, obesity is a recognized risk factor forincreased incidence of complications of general anesthesia. (See e. g.,Kopelman, Nature 404:635-43, 2000). In general, obesity reduces lifespan and carries a serious risk of co-morbidities such as those listedabove.

Other diseases or disorders associated with obesity are birth defects,maternal obesity being associated with increased incidence of neuraltube defects, carpal tunnel syndrome (CTS); chronic venous insufficiency(CVI); daytime sleepiness; deep vein thrombosis (DVT); end stage renaldisease (ESRD); gout; heat disorders; impaired immune response; impairedrespiratory function; infertility; liver disease; lower back pain;obstetric and gynecologic complications; pancreatitis; as well asabdominal hernias; acanthosis nigricans; endocrine abnormalities;chronic hypoxia and hypercapnia; dermatological effects; elephantitis;gastroesophageal reflux; heel spurs; lower extremity edema; mammegalywhich causes considerable problems such as bra strap pain, skin damage,cervical pain, chronic odors and infections in the skin folds under thebreasts, etc.; large anterior abdominal wall masses, for exampleabdominal panniculitis with frequent panniculitis, impeding walking,causing frequent infections, odors, clothing difficulties, low backpain; musculoskeletal disease; pseudo tumor cerebri (or benignintracranial hypertension), and sliding hiatil hernia.

The present invention further provides a method for increasing energyexpenditure in a subject. The method includes, for example, peripherallyadministering a therapeutically effective amount of a PYY analogue ofthe invention to the subject, thereby altering energy expenditure.Energy is burned in all physiological processes. The body can alter therate of energy expenditure directly, by modulating the efficiency ofthose processes, or changing the number and nature of processes that areoccurring. For example, during digestion the body expends energy movingfood through the bowel, and digesting food, and within cells, theefficiency of cellular metabolism can be altered to produce more or lessheat.

In one aspect, the method of the invention involves manipulation of thearcuate circuitry that alter food intake coordinately and reciprocallyalter energy expenditure. Energy expenditure is a result of cellularmetabolism, protein synthesis, metabolic rate, and calorie utilization.Thus, in this aspect of the invention, administration of a PYY analogueaccording to the invention results in increased energy expenditure, anddecreased efficiency of calorie utilization.

The invention also provides a method for improving a lipid profile in asubject comprising administration of a PYY analogue according to theinvention, or a pharmaceutical composition comprising the PYY analogue,to the subject. The invention also provides a method for alleviating acondition or disorder that can be alleviated by reducing nutrientavailability, comprising administration of a PYY analogue according tothe invention, or a pharmaceutical composition comprising the PYYanalogue, to the subject.

Appetite can be measured by any means known to one of skill in the art.For example, decreased appetite can be assessed by a psychologicalassessment. For example, administration of a compound of the inventionresults in a change in perceived hunger, satiety, and/or fullness.Hunger can be assessed by any means known to one of skill in the art.For example, hunger is assessed using psychological assays, such as byan assessment of hunger feelings and sensory perception using aquestionnaire, such as, but not limited to, a Visual Analog Score (VAS)questionnaire. In one specific, non-limiting example, hunger is assessedby answering questions relating to desire for food, drink, prospectivefood consumption, nausea, and perceptions relating to smell or taste.

A PYY analogue of the invention may be used for weight control andtreatment, for example reduction or prevention of obesity, in particularany one or more of the following: preventing and reducing weight gain;inducing and promoting weight loss; and reducing obesity as measured bythe Body Mass Index. A PYY analogue of the invention may be used in thecontrol of any one or more of appetite, satiety and hunger, inparticular any one or more of the following: reducing, suppressing andinhibiting appetite; inducing, increasing, enhancing and promotingsatiety and sensations of satiety; and reducing, inhibiting andsuppressing hunger and sensations of hunger. A PYY analogue of theinvention may be used in maintaining any one or more of a desired bodyweight, a desired Body Mass Index, a desired appearance and good health.Accordingly, the invention also provides a method of causing weight lossor preventing weight gain in a subject for cosmetic purposes, comprisingadministering an effective amount of an analogue of PYY according to theinvention, or a composition comprising the PYY analogue, to the subject.

A subject may be a subject who desires weight loss, for example femaleand male subjects who desire a change in their appearance. A subject maydesire decreased feelings of hunger, for example the subject may be aperson involved in a lengthy task that requires a high level ofconcentration, for example soldiers on active duty, air trafficcontrollers, or truck drivers on long distance routes, etc.

The present invention may also be used in treating, prevention,ameliorating or alleviating conditions or disorders caused by,complicated by, or aggravated by a relatively high nutrientavailability. The term “condition or disorder which can be alleviated byreducing caloric (or nutrient) availability” is used herein to denoteany condition or disorder in a subject that is either caused by,complicated by, or aggravated by a relatively high nutrientavailability, or that can be alleviated by reducing nutrientavailability, for example by decreasing food intake. Subjects who areinsulin resistant, glucose intolerant, or have any form of diabetesmellitus, for example, type 1, 2 or gestational diabetes, can alsobenefit from methods in accordance with the present invention.

The invention relates to the treatment of metabolic disorders, forexample disorders of energy metabolism. Such disorders includeconditions or disorders associated with increased caloric intakeinclude, but are not limited to, insulin resistance, glucoseintolerance, obesity, diabetes, including type-2 diabetes, eatingdisorders, insulin-resistance syndromes, and Alzheimer’s disease.

According to the present invention, the PYY analogue is preferably usedin the treatment of a human. However, while the compounds of theinvention will typically be used to treat human subjects they may alsobe used to treat similar or identical conditions in other vertebratesfor example other primates; farm animals for example swine, cattle andpoultry; sport animals for example horses; companion animals for exampledogs and cats.

Compositions

While it is possible for the active ingredient to be administered alone,it is preferable for it to be present in a pharmaceutical formulation orcomposition. Accordingly, the invention also provides a pharmaceuticalcomposition comprising an analogue of PYY according to the inventiontogether with a pharmaceutically acceptable carrier and optionally othertherapeutic ingredients. Pharmaceutical compositions of the inventionmay take the form of a pharmaceutical formulation as described below.

The pharmaceutical formulations according to the invention include thosesuitable for oral, parenteral (including subcutaneous, intradermal,intramuscular, intravenous, and intraarticular), inhalation (includingfine particle dusts or mists which may be generated by means of varioustypes of metered dose pressurized aerosols, nebulizers or insufflators),rectal and topical (including dermal, transdermal, transmucosal, buccal,sublingual, and intraocular) administration, although the most suitableroute may depend upon, for example, the condition and disorder of therecipient.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, sachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Various pharmaceuticallyacceptable carriers and their formulation are described in standardformulation treatises, e.g., Remington’s Pharmaceutical Sciences by E.W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of ParenteralScience and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein. The present compounds can, for example, beadministered in a form suitable for immediate release or extendedrelease. Immediate release or extended release can be achieved by theuse of suitable pharmaceutical compositions comprising the presentcompounds, or, particularly in the case of extended release, by the useof devices such as subcutaneous implants or osmotic pumps. The presentcompounds can also be administered liposomally.

Preferably, compositions according to the invention are suitable forsubcutaneous administration, for example by injection. According tocertain embodiments the composition may contain metal ion for examplecopper, iron, aluminium, zinc, nickel or cobalt ions. The presence ofsuch ions may limit solubility and thus delay absorption into thecirculatory system from the site of subcutaneous administration. In aparticularly preferred embodiment, the composition contains zinc ions.Zinc ions may be present at any suitable concentration for example at amolar ratio to peptide molecules of 10:1 to 1:10, 8:1 to 1:8, 5:1 to1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2 or 1:1. In one embodiment, thepharmaceutical composition has a pH of less than 5 and thepharmaceutical composition comprises zinc ions.

Exemplary compositions for oral administration include suspensions whichcan contain, for example, microcrystalline cellulose for imparting bulk,alginic acid or sodium alginate as a suspending agent, methylcelluloseas a viscosity enhancer, and sweeteners or flavoring agents such asthose known in the art; and immediate release tablets which can contain,for example, microcrystalline cellulose, dicalcium phosphate, starch,magnesium stearate and/or lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants such as those known inthe art. PYY analogues of the invention or variants, derivatives, saltsor solvates thereof can also be delivered through the oral cavity bysublingual and/or buccal administration. Molded tablets, compressedtablets or freeze-dried tablets are exemplary forms which may be used.Exemplary compositions include those formulating the present compound(s)with fast dissolving diluents such as mannitol, lactose, sucrose and/orcyclodextrins. Also included in such formulations may be high molecularweight excipients such as celluloses (avicel) or polyethylene glycols(PEG). Such formulations can also include an excipient to aid mucosaladhesion such as hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleicanhydride copolymer (e.g., Gantrez), and agents to control release suchas polyacrylic copolymer (e.g. Carbopol 934). Lubricants, glidants,flavors, coloring agents and stabilizers may also be added for ease offabrication and use.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example saline or water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described. Exemplary compositions for parenteraladministration include injectable solutions or suspensions which cancontain, for example, suitable non-toxic, parenterally acceptablediluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer’ssolution, an isotonic sodium chloride solution, or other suitabledispersing or wetting and suspending agents, including synthetic mono-or diglycerides, and fatty acids, including oleic acid, or Cremaphor. Anaqueous carrier may be, for example, an isotonic buffer solution at a pHof from about 3.0 to about 8.0, preferably at a pH of from about 3.5 toabout 7.4, for example from 3.5 to 6.0, for example from 3.5 to about5.0. Useful buffers include sodium citrate-citric acid and sodiumphosphate-phosphoric acid, and sodium acetate/acetic acid buffers. Thecomposition preferably does not include oxidizing agents and othercompounds that are known to be deleterious to PYY and related molecules.Excipients that can be included are, for instance, other proteins, suchas human serum albumin or plasma preparations. If desired, thepharmaceutical composition may also contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

In one embodiment, the pharmaceutical composition is present in asyringe or other administration device for subcutaneous administrationto humans.

Exemplary compositions for nasal aerosol or inhalation administrationinclude solutions in saline, which can contain, for example, benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, and/or other solubilizing or dispersing agents such asthose known in the art. Conveniently in compositions for nasal aerosolor inhalation administration the compound of the invention is deliveredin the form of an aerosol spray presentation from a pressurized pack ora nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoro-methane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator can be formulatedto contain a powder mix of the compound and a suitable powder base, forexample lactose or starch. In one specific, non-limiting example, acompound of the invention is administered as an aerosol from a metereddose valve, through an aerosol adapter also known as an actuator.Optionally, a stabilizer is also included, and/or porous particles fordeep lung delivery are included (e.g., see U.S. Pat. No. 6,447,743).

Formulations for rectal administration may be presented as a retentionenema or a suppository with the usual carriers such as cocoa butter,synthetic glyceride esters or polyethylene glycol. Such carriers aretypically solid at ordinary temperatures, but liquefy and/or dissolve inthe rectal cavity to release the drug.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavoured basis such as sucrose and acacia ortragacanth, and pastilles comprising the active ingredient in a basissuch as gelatin and glycerine or sucrose and acacia. Exemplarycompositions for topical administration include a topical carrier suchas Plastibase (mineral oil gelled with polyethylene).

Preferred unit dosage formulations are those containing an effectivedose, as hereinbefore recited, or an appropriate fraction thereof, ofthe PYY analogue.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The PYY analogues of the invention are also suitably administered assustained-release systems. Suitable examples of sustained-releasesystems of the invention include suitable polymeric materials, forexample semi-permeable polymer matrices in the form of shaped articles,e.g., films, or mirocapsules; suitable hydrophobic materials, forexample as an emulsion in an acceptable oil; or ion exchange resins; andsparingly soluble derivatives of the compound of the invention, forexample, a sparingly soluble salt. Sustained-release systems may beadministered orally; rectally; parenterally; intracistemally;intravaginally; intraperitoneally; topically, for example as a powder,ointment, gel, drop or transdermal patch; bucally; or as an oral ornasal spray.

Preparations for administration can be suitably formulated to givecontrolled release of compounds of the invention. For example, thepharmaceutical compositions may be in the form of particles comprisingone or more of biodegradable polymers, polysaccharide jellifying and/orbioadhesive polymers, amphiphilic polymers, agents capable of modifyingthe interface properties of the particles of the compound of formula(I). These compositions exhibit certain biocompatibility features whichallow a controlled release of the active substance. See U.S. Pat. No.5,700,486.

A PYY analogue of the invention may be delivered by way of a pump (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201, 1987;Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med.321:574, 1989) or by a continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed. Thekey factor in selecting an appropriate dose is the result obtained, asmeasured by decreases in total body weight or ratio of fat to lean mass,or by other criteria for measuring control or prevention of obesity orprevention of obesity-related conditions, as are deemed appropriate bythe practitioner. Other controlled release systems are discussed in thereview by Langer (Science 249:1527-1533, 1990). In another aspect of thedisclosure, compounds of the invention are delivered by way of animplanted pump, described, for example, in U.S. Pat. No. 6,436,091; U.S.Pat. No. 5,939,380; U.S. Pat. No. 5,993,414.

Implantable drug infusion devices are used to provide patients with aconstant and long term dosage or infusion of a drug or any othertherapeutic agent. Essentially such device may be categorized as eitheractive or passive. A compound of the present invention may be formulatedas a depot preparation. Such a long acting depot formulation can beadministered by implantation, for example subcutaneously orintramuscularly; or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials, for example as an emulsion in an acceptable oil; or ionexchange resins; or as a sparingly soluble derivatives, for example, asa sparingly soluble salt.

A therapeutically effective amount of a PYY analogue of the inventionmay be administered as a single pulse dose, as a bolus dose, or as pulsedoses administered over time. Thus, in pulse doses, a bolusadministration of a PYY analogue of the invention is provided, followedby a time period wherein no a compound of the invention is administeredto the subject, followed by a second bolus administration. In specific,non-limiting examples, pulse doses of a compound of the invention areadministered during the course of a day, during the course of a week, orduring the course of a month.

The invention also provides an analogue of PYY according to theinvention together with a further therapeutic agent, for simultaneous,sequential or separate administration. The invention also provides apharmaceutical composition comprising the PYY analogue according to theinvention and a further therapeutic agent. Examples of furthertherapeutic agents include an additional appetite suppressant, afood-intake-reducing, plasma glucose-lowering or plasma lipid-alteringagent. Specific, non-limiting examples of an additional appetitesuppressant include amfepramone (diethylpropion), phentermine, mazindoland phenylpropanolamine, fenfluramine, dexfenfluramine, and fluoxetine.As mentioned above, the PYY analogue of the invention can beadministered simultaneously with the additional appetite suppressant, orit may be administered sequentially or separately. In one embodiment,the compound of the invention is formulated and administered with anappetite suppressant in a single dose.

A PYY analogue of the invention may be administered whenever the effect,e.g., appetite suppression, decreased food intake, or decreased caloricintake, is desired, or slightly before to whenever the effect isdesired, such as, but not limited to about 10 minutes, about 15 minutes,about 30 minutes, about 60 minutes, about 90 minutes, or about 120minutes, before the time the effect is desired.

The therapeutically effective amount of a PYY analogue of the inventionwill be dependent on the molecule utilized, the subject being treated,the severity and type of the affliction, and the manner and route ofadministration. For example, a therapeutically effective amount of a PYYanalogue of the invention may vary from about 0.01 µg per kilogram (kg)body weight to about 1 g per kg body weight, for example about 0.1 µg toabout 20 mg per kg body weight, for example about 1 µg to about 5 mg perkg body weight, or about 5 µg to about 1 mg per kg body weight.

In one embodiment of the invention, a PYY analogue of the invention maybe administered to a subject at from 5 to 1000 nmol per kg bodyweight,for example at from 10 to 750 nmol per kg bodyweight, for example atfrom 20 to 500 nmol per kg bodyweight, in particular at from 30 to 240nmol per kg bodyweight. For a 75 kg subject, such doses correspond todosages of from 375 nmol to 75 µmol, for example from 750 nmol to 56.25µmol, for example from 1.5 to 37.5 µmol, in particular from 2.25 to 18µmol.

In an alternative embodiment, a PYY analogue of the invention may beadministered to a subject at 0.5 to 135 picomole (pmol) per kg bodyweight, for example 5 to 100 picomole (pmol) per kg body weight, forexample 10 to 90 picomole (pmol) per kg body weight, for example about72 pmol per kg body weight. In one specific, non-limiting example, a PYYanalogue of the invention is administered in a dose of about 1 nmol ormore, 2 nmol or more, or 5 nmol or more. In this example, the dose ofthe PYY analogue of the invention is generally not more than 100 nmol,for example, the dose is 90 nmols or less, 80 nmols or less, 70 nmols orless, 60 nmols or less, 50 nmols or less, 40 nmols or less, 30 nmols orless, 20 nmols or less, 10 nmols. For example, a dosage range maycomprise any combination of any of the specified lower dose limits withany of the specified upper dose limits. Thus, examples of non-limitingdose ranges of compounds of the invention are within the range of from 1to 100 nmols, from 2 to 90 mols, from 5 to 80 nmols.

In one specific, non-limiting example, from about 1 to about 50 nmol ofa PYY analogue of the invention is administered, for example about 2 toabout 20 nmol, for example about 10 nmol is administered as asubcutaneous injection. The exact dose is readily determined by one ofskill in the art based on the potency of the specific PYY analogueutilized, the route of delivery of the PYY analogue and the age, weight,sex and physiological condition of the subject.

Suitable doses of PYY analogue of the invention also include those thatresult in a reduction in calorie intake, food intake, or appetite, orincrease in energy expenditure that is equivalent to the reduction incalorie intake, food intake, or appetite, or to increase the energyexpenditure, caused by the normal postprandial level of PYY. Examples ofdoses include, but are not limited to doses that produce the effectdemonstrated when the serum levels of PYY are from about 40 pM to about60 pM, or from about 40 pM to about 45 pM, or about 43 pM.

The doses discussed above may be given, for example, once, twice,three-times or four-times a day. Alternatively, they may be give onceevery 2, 3 or 4 days. In a slow release formulation containing zinc, itmay be possible to give a dose once every 3, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 days. According to certain embodimentsthey may be administered once shortly before each meal to be taken.

Specific Sequences of the Invention

According to certain specific embodiments of the invention the analogueof PYY has an amino acid sequence given in one of the specific sequencesset out in FIGS. 1A-1D.

The invention is illustrated by the following non-limiting Examples.

EXAMPLES Materials and Methods Peptide Synthesis

Peptides were made by a standard automated fluorenylmethoxycarbonyl(Fmoc) solid phase peptide synthesis (SPPS) method. Peptide synthesiswas carried out on a tricyclic amide linker resin. Amino acids wereattached using the Fmoc strategy. Each amino acid was added sequentiallyfrom the C— to the N-termini. Peptide couplings were mediated by thereagent TBTU. Peptide cleavage from the resin was achieved withtrifluoracetic acid in the presence of scavengers. Native PYY 3-36 NH₂is obtained as described previously (WO03/026591); de novo synthesisusing tricyclic amide resin and Fmoc chemistry is also possible.

Peptides were purified by reverse phase HPLC. Full quality control wasperformed on all purified peptides and peptides were shown to be greaterthan 95% pure by HPLC in two buffer systems. Amino acid analysisfollowing acid hydrolysis confirmed the amino acid composition. MALDI-MSshowed the expected molecular ion.

Binding Studies

Membrane preparation of HEK 293 cells overexpressing the human Y2receptor (NPYR200000, Missouri S&T cDNA resource center) were isolatedby osmotic lysis and differential centrifugation as described by Morganet al (Neuroendocrinol 1996. 8 283-290). Receptor binding assays werecompleted as described by Druce et al (2009 Endocrinology 150(4) 712-22)except the buffer used was 0.02 M HEPES pH 7.4, 5 mM CaCl₂, 1 mM MgCl₂,1% bovine serum albumin, 0.1 mM diprotin A, 0.2 mM PMSF, 10 µMphosphoramidon, ¹²⁵I-PYY₁₋₃₆ as the radiolabel and the human Y2 receptorused.

In Vitro Receptor Potency Studies

DiscoverX® hY2 CHO-K1 cells (10,000 cells per well (96 well plate)) wereresuspended in media containing 0.2 mM IBMX and 0.02 mM forskolin (whichstimulates cAMP production) and test peptides at a range ofconcentrations, for 30 minutes. The reaction stopped by lysing the cellsand cAMP quantified 60 minutes later using Cisbio cAMP dynamic 2 kit.The peptides tested were “Y1419” falling within the scope of the presentinvention and having the sequencePro—Ile—Lys—Pro—Glu—Ala—Pro—Gly—Glu—Gly—Ala—Ser—Pro—Glu—Glu—Leu—Leu—His—Tyr—Tyr—Ala—Ala—Leu—Arg—His—Phe—Leu—Asn—His—Val—Thr—Arg—Gln—Arg—Tyr—NH₂(SEQ ID No—23) and a comparator peptide “Y242” having the sequence

Pro-Ile-His-Pro-His-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-His-Tyr-Tyr-Ala-Ala-Leu-Arg-His-Tyr-Leu-Asn-His-Val-Thr-Arg-Gln-Arg-Tyr-NH₂ (SEQ ID NO: 44).

Animals

Male Wistar rats (Charles River Ltd, Margate, UK) were used for animalexperiments.

Feeding Studies in Rats

Rats were individually housed in IVC cages. Animals were randomised intotreatment groups, with stratification by body weight. All peptidesolutions were prepared freshly immediately prior to administration. Thevehicle used for all studies was 5% v/v water and 95% NaCl (0.9% w/v).Peptide and vehicle were administered by subcutaneous injection with adosage of either 25 or 50 nM/Kg body weight. Animals were injected every24 hours over 6 days and the study continued for a further 24 hoursgiving 7 days total from the first injection to the end of the study.Animals were given free access to food and water during the studyperiod. Animals were weighed at the end of the study and total weightloss, if any, was recorded.

Results

FIG. 1A discloses a number of specific sequences encompassed by thescope of the present invention in all its aspects. Each of thesesequences is a specific embodiment of the invention. The numberallocated to each sequence corresponds to the “Y” number referred toelsewhere herein (ie the line beginning with “1419” is understood todisclose the sequence of analogue number “Y1419”) It also discloses, onthe first line, the sequence of naturally occurring human PYY forreference.

The table below provides receptor potency data for the example peptidesof FIGS. 1A-1D at the human Neuropeptide Y Receptor Y2 overexpressed inhY2 CHO-K1 cells (DiscoverX®). Ten thousand cells per well (96 wellplate) were resuspended in media containing 0.2 mM IBMX and 0.02 mMforskolin (which stimulates cAMP production) and test peptides at arange of concentrations, for 30 minutes. The reaction stopped by lysingthe cells and cAMP quantified 60 minutes later using Cisbio cAMP dynamic2 kit. The values provided are the ratios of the example compound EC₅₀to the PYY₂₋₃₆ EC₅₀ (e.g. for the “Average ratio to PYY₂₋₃₆ ± SEM”column, a value of 0.5 would indicate that the concentration of theexample compound required to inhibit 50% maximum release of cAMP is halfthe concentration of PYY₂₋₃₆ that is required, and a value of 2 wouldindicate that the concentration of the example compound required toinhibit 50% maximum release of cAMP is 2 times that of PYY₂₋₃₆ . It canbe seen that example peptides of the invention are much more potentinhibitor than the previous generation of preferred compound (Y242included in the table for comparative purposes).

Peptide Average ratio to PVV₂₋₃₆ ± SEM Y242 10.0 ± 0.69 Y1276 0.6 ± 0.06Y1319 0.6 ± 0.09 Y1371 0.9 ± 0.06 Y1372 1.1 ± 0.16 Y1377 1.0 ± 0.07Y1379 0.9 ± 0.09 Y1419 0.8 ± 0.16 Y1421 0.9 ± 0.09 Y1431 0.9 ± 0.07Y1447 1.4 ± 0.31 Y1448 1.7 ± 0.46 Y1450 1.1 ± 0.20 Y1477 0.9 ± 0.06Y1489 1.0 ± 0.06 Y1490 0.9 ± 0.28 Y1518 1.7 ± 0.13 Y1528 0.5 ± 0.08Y1553 0.8 ± 0.25 Y1558 0.6 ± 0.05

FIG. 2 shows the differential ability of compound Y242 and compoundY1419 to inhibit forskolin production. It can be seen that 1419 is muchmore potent inhibitor than the previous generation of preferredcompounds.

FIGS. 3A-3D and FIGS. 4A-4D show the results of rat feeding experimentscomparing the activity of Y242 (FIGS. 3A-3D) and Y1419 (FIGS. 4A-4D) inovernight fasted male Wistar rats which have received a singlesubcutaneous injection of control saline or peptide in saline. Theirfood intake and body weight are monitored for the following 6 days andthe values shown are the mean +/- SEM. It can be seen that although thecomparator compound Y242 produced promising reductions in food intakeand body weight for the first 48 hours, that reduction is not sustainedfor the longer time period (suggestion that the comparator compoundalthough efficacious would need to be re-dosed after a day or two).Compound Y1419 performs much better and a single initial dose is stillactive at 160 hours after administration suggesting that the compound ispromising for use as a medicine which would be administered weekly orless often.

FIGS. 5A-5D show the results of feeding experiments wherein fasted (24h) male Wistar rats (Charles River) received a single subcutaneousinjections of saline or example peptide of the invention (100 nmol/kg ).Total food intake from dosing to 48 h after dosing was measured, as wasbody weight change from pre-fasted to 48 h post dose.

FIGS. 6A-6T show the ability of various indicated compounds of theinvention to inhibit forskolin production. Data for PYY 2-36 is alsoincluded as a comparator.

1. A method of preventing or treating a disorder of energy metabolismsuch as diabetes and/or obesity, preventing loss of pancreatic isletfunction and/or recovering pancreatic islet function, reducing appetite,reducing food intake, and/or reducing calorie intake in a subject,comprising administering to the subject a therapeutically effectiveamount of an analogue of PYY or a derivative thereof which differs fromthe sequence of native human PYY (SEQ ID NO: 1) in the followingrespects: i. Ser23 is substituted with Ala23, Glu23, or Aib23, ii. Asn18is substituted with Leu18, Aib18 or A1a18,and iii. the analogue of PYYfurther differs from the sequence of native human PYY in at least one ofthe following respects: Tyrl is absent, Glu10 is substituted with Lys10or G1n10, Asp11 is substituted with Gly11, Arg19 is substituted withHis19, Tyr 27 is substituted with Phe27, Leu30 is substituted withHis30, and Val31 is substituted with Leu31, wherein the C-terminalresidue optionally terminates in a primary amide group (—C(O)NH₂) inplace of a carboxylic acid group (—CO₂H); and wherein the derivative isselected from the group consisting of glycosylation, carbamylation,acylation, sulfation, phosphorylation, cyclization, lipidization,pegylation or fusion to another peptide or protein to form a fusionprotein or a salt or solvate of the analogue or the derivative of theanalogue.
 2. The method as claimed in claim 1, wherein the C-terminalresidue terminates in a primary amide group (—C(O)NH₂) in place of acarboxylic acid group (—CO₂H).
 3. The method as claimed in claim 1,wherein Ser23 is substituted with Ala23.
 4. A method of preventing ortreating a disorder of energy metabolism such as diabetes and/orobesity, preventing loss of pancreatic islet function and/or recoveringpancreatic islet function, reducing appetite, reducing food intake,and/or reducing calorie intake in a subject, comprising administering tothe subject a therapeutically effective amount of an analogue of PYY oraderivative thereof, wherein the analogue of PYY is selected from thegroup consisting of Y1372 (SEQ IDNO: 20), Y1419 (SEQ ID NO: 23), Y1421(SEQ ID NO: 24), Y1518 (SEQ IDNO: 32), Y1528 (SEQ ID NO: 33), Y1558 (SEQIDNO: 35), Y1568 (SEQ ID NO: 36), and Y1579 (SEQ ID NO: 41), wherein thederivative is selected from the group consisting of amidation,glycosylation, carbamylation, acylation, sulfation, phosphorylation,cyclization, lipidization, pegylation or fusion to another peptide orprotein to form a fusion protein; or a salt or solvate of the analogueor the derivative of the analogue.
 5. The method as claimed in claim 4,wherein the analogue of PYY is compound Y1419 (SEQ ID NO: 23).
 6. Themethod as claimed in claim 5, wherein the disorder of energy metabolismis diabetes and/or obesity.
 7. The method as claimed in claim 1, whereinGlu10 is unsubstituted, Asn18 is substituted with Leu18, and Ser23 issubstituted with Ala23.
 8. The method as claimed in claim 1, whereinAsn18 is substituted with Leu18, and Val31 is unsubstituted.
 9. A methodas claimed in claim 1 wherein the method comprises administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising the analogue of PYY or derivative thereoftogether with a pharmaceutically acceptable carrier and optionally afurther therapeutic agent.
 10. The method as claimed in claim 9, whereinthe further therapeutic agent is selected from an analogue of GLP-1, ananalogue of glucagon, or a derivative of either thereof.
 11. The methodas claimed in claim 9, wherein the pharmaceutical composition furthercomprises Zn²⁺ions.
 12. The method as claimed in claim 1, wherein thedisorder of energy metabolism is diabetes and/or obesity.
 13. The methodas claimed in claim 12, wherein the analogue of PYY is compound Y1419(SEQ ID NO: 23).
 14. The method as claimed in claim 1, wherein thesubject is overweight, obese and/or diabetic.
 15. The method as claimedin claim 13, wherein the subject is diabetic, for example the subjecthas Type II diabetes, or the subject has Type I diabetes.
 16. The methodas claimed in claim 1, wherein the therapeutically effective amount ofan analogue of PYY or a derivative thereof is administered parenterally.17. The method as claimed in claim 1, wherein the therapeuticallyeffective amount of an analogue of PYY or a derivative thereof isadministered subcutaneously.
 18. A method of causing weight loss orpreventing weight gain in a subject for cosmetic purposes, comprisingadministering to a subject an effective amount of an analogue of PYY orderivative thereof which differs from the sequence of native human PYY(SEQ ID NO: 1) in the following respects: i. Ser23 is substituted withAla23, Glu23, or Aib23, ii. Asn18 is substituted with Leu18, Aib18 orA1a18, and iii. the analogue of PYY further differs from the sequence ofnative human PYY in at least one of the following respects: Tyrl isabsent, Glu10 is substituted with Lys10 or G1n10, Asp11 is substitutedwith Gly11, Arg19 is substituted with His19, Tyr 27 is substituted withPhe27, Leu30 is substituted with His30, and Val31 is substituted withLeu31, wherein the C-terminal residue optionally terminates in a primaryamide group (—C(O)NH₂) in place of a carboxylic acid group (—CO₂H); andwherein the derivative is selected from the group consisting ofglycosylation, carbamylation, acylation, sulfation, phosphorylation,cyclization, lipidization, pegylation or fusion to another peptide orprotein to form a fusion protein or a salt or solvate of the analogue orthe derivative of the analogue; or comprising administering atherapeutically effective amount of a pharmaceutical compositioncomprising the analogue of PYY together with a pharmaceuticallyacceptable carrier and optionally a further therapeutic agent.
 19. Themethod as claimed in claim 18, wherein the C-terminal residue terminatesin a primary amide group (—C(O)NH₂) in place of a carboxylic acid group(—CO₂H).
 20. The method as claimed in claim 18, wherein Ser23 issubstituted with Ala23.